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Domain organization of eukaryotic genome
Cell Biology lntemational Reports, VoL 16, No. 8, 1992
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DOMAIN ORGANIZATION OF EUKARYOTIC GENOME
S.V. Razin and Y.S. Vassetzky Institute of Biology of the Gene, USSR Academy of Sciences, 34/5 Vavilov st., Moscow 117334, USSR
INTRODUCTION It is a well-established fact that transcription of eukaryotic genes is regulated at several levels. One of these levels is represented by mechanisms that operate within relatively large genomic domains. Active domains are preferentially sensitive to DNase I (for review see Gross and Garrard, 1988). They could be several dozens kb long and usually include a group of functionally related genes (e.g. globin genes). The borders of the domains are quite sharp. Recent fmdings indicate that transcriptional status of a whole domain can be determined by a so-called dominant control region (DCR). The best known example is the dominant control region of the domain of human ~-globin genes (Grosveld et al., 1987; Groodin et al., 1989). A relatively short region located in the upstream area of this domain drastically influences the functional status of a genomic region that is more than 100 Kbp long (in particular: transcriptional status of this region, sensitivity to DNase I, methylation pattern and replication thning). Existence of functional domains in eukaryotic genome raises a question whether these domains correspond to some stmctural units in chromatin. There are at least three levels of DNA packaging in chromatin, namely nucleosomal level, solenoid level and DNA loops level (for review see Reeves, 1984). Only the structural units of the third level of DNA compactization (i.e. DNA loops anchored at the chromosomal skeleton) have the size comparable with the size of the above discussed functional genomic domains. The DNA loops were first recognized when sedimentation properties of high-salt extracted nuclei were studied (Cook and BrazeU, 1980). It turned out that after extraction of histones the DNA was topologically constrained and could be either supercofled or relaxed depending on some external parameters. The average size of constrained domains determined by several independent methods was found to be of the order of 100 kb (for review see Hancock and Huges, 1982). An attractive hypothesis has been suggested that individual loops correspond to the functional genomic domains (Cook, 1989) In the frame of this hypothesis it was easy to explain how the preferential sensitivity to DNase I could be maintained within the limited though large areas of the genome and why the borders of the sensitive regions are rather sharp. The most simple suggestion appeals to the possibility of reversible distraction of solenoid level of DNP packaging within the individual loops. All the above mentioned considerations draw particular attention to the problem of sequence specificity of DNA interaction with the chromosomal skeletal elements. Indeed, if loops correspond to functional genomic domains, one could expect that borders of the loops have specific positions on the DNA chain and and are located 0309-1651/92/080697-12/$03.00/0
© 1992 Academic PressLtd
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between the functional units of the genome. We shall discuss the results of experiments aimed to check this predictions. SEQUENCE SPECIFICITY OF DNA INTERACTIONWITH THE NUCLEAR(CHROMOSOMAL) SKELETON REVEALED IN THE EXPERIMENTS UTIIJZING HIGH SALT EXTRACTION PROTOCOL OF NUCLEAR MATRIX ISOLATION
The important feature of large DNA loops attached to the chromosomal scaffold that should be outlined here is the resistance of the whole structure to the extraction with concentrated salt solution. The remarkable stability of DNA interaction with chromosomal scaffold (that in interphase nuclei constitutes part of more complex structure known as nuclear matrix; Berezney and Coffey, 1976) opened a simple possibility of isolation of DNA fragments located at basements of chromatin loops. For isolation of loop ends one simply has to preextract nuclei with concentrated or medium salt solution, than to introduce double-stranded breaks into DNA chain (limited digestion with restriction enzymes or limited digestion with sequence-nonspecific nucleases) and finally to separate scaffold-bound and cleaved-off DNA by several washes with concentrated salt solution (Cook and Brazell, 1980; Pardoll and Vogelstein, 1980; Razin et al., 1979). Different modifications of this approach were widely used for isolation of the so-called nuclear matrix DNA (nmDNA). The latter can be defined as a preparation of DNA fragments that remains bound to the residual nuclear structures after high-salt extraction of nuclease-treated nuclei. The variations in experimental procedures used by different authors usually involved utilization of different salts (NaCI, KCI, (NH4)2SO4, etc.) and different concentrations of salt for preextraction and final extraction as well as utilization of different enzymes for DNA cleavage (for review see Razin, 1987). After isolation of nmDNA, one obviously tried to answer two distinct, though related questions: I. Do the loop ends have specific positions in the genome? and II. Are there specific DNA sequences located at the basements of DNA loops? In our initial experiments (Razin et al., 1978, 1979) the renaturation properties of nmDNA were studied. This DNA was found to be moderately enriched in repetitive sequences. This observation was later confumed by some authors (Hancock and Huges, 1982; Jackson et al., 1984; Matsumoto, 1981) while the others did not observe any specificity of runDNA (Basler et al., 1981). Possible reasons of these contradictions have been discussed previously (Razin et al., 1985; Razin, 1987). More disappointing, no particular DNA anchoring sequence was found either in our experiments or in experiments of other authors. Even though enriched in repetitive sequences, the nmDNA still contained a significant portion of unique sequences. The spectrum of repeats represented in nmDNA was also rather broad. More meaningful results were obtained in the experiments ahned to find the relative positions of different genes within DN.A loops. These experiments initially designed by Cook and Brazell (1980) were later carded out in a number of laboratories. In most of the papers published so far, a correlation between the transcriptional status of a gene and its attaclmaent to the nuclear matrix was observed. The active genes were generally found in the nmDNA while the non-active ones in the cleaved off fraction (for review see Razin, 1987). The most convincing results
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were probably obtained in the experiments with hormone-induced premature cell differentiation. The cell type specific genes that started expression in differentiated cells were associated with the nuclear matrix in these cells and were not associated with the nuclear matrix in maternal (non-differentiated) cells.
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Fig. 1. Detection of specific regions in the chicken tx-globin domain that interact with erythrocyte and erythroblast nmDNA (Razin et al., 1985). Left panel: identification of attachment sites in chicken erythroblast nuclei. Restriction fragments of recombinant clones were transferred onto nylon filters and hybridized with either nick-translated total DNA (a-f) or erythroblast nmDNA probe (a'-f'). The scheme below indicates the regions of the domain of chicken 0t-globin genes that are highly (black) or moderately (shaded) enriched in nmDNA as compared to total DNA. Circles-filled areas correspond to repetitive sequences. Arrows indicate recognition sites for restriction endonucleases used. Right panel: identification of attachment sites in chicken erythrocyte nuclei. Restriction fragments of recombinant clones were transferred onto nylon filters and hybridized with either nick-translated total DNA (a-e) or erythrocyte nmDNA probe (a'-e'). The scheme is similar to that in the left panel. The scheme below the panels summarizes the data on distribution of attachment sites in the both ceil types.
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The positive correlation between expression of viral genomes integrated into the genome of host cells and their attachment to the nuclear matrix of these cells was also reported (Cook et al., 1982). When the large cloned areas of the eukaryotic genome became available, the positions of preferential sites of DNA interaction with the nuclear matrix have been mapped within several such areas (Razin et al., 1986, 1990) and the positive correlation between DNA attachment to the nuclear matrix mad its transcription has also been observed. All the above-mentioned results could h a r d y be assimilated by the initial model based on the assumption that there was a correlation between DNA loop organization and domain organization of the genome. Indeed, all the specific interactions of DNA with nuclear matrix seemed to be related to some functional processes and patterns of these interactions were different in the cells of different lineages. At the same time a new model of DNA loop organization based on function-dependent association of DNA with the nuclear matrix also could not explain all the experimental data, for example a correlation of DNA loops size with the average sizes of replicons in the cells of different species (Buongiomo et al., 1982). Several years ago we suggested that there is a special group of "structural" attachment sites that are different from transcription-related attachment sites and that the former defined the borders of genomic domains. To check the idea we carried out experiments with inactive nuclei, e.g. mature sperm nuclei or repressed nuclei of avian erythrocytes. In these nuclei, all the function-dependent interactions of DNA with the nuclear matrix should disappear and "structural" interactions, should be easily visible. We have mapped preferential positions of sites of DNA attachment to the nuclear matrix in the domain of chicken ct-globin genes in chicken erythroblasts, erythrocytes and mature sperm nuclei. The results of our experiments (Fig. 1) supported the initial suggestion. While in erythroblast nuclei the whole transcriptionally active area was attached to the nuclear matrix, in erythrocyte and sperm nuclei only one major attachment site was found in the upstream region of the domain (Razin et al., 1985; Razin, 1987; Kalandadze et al., 1990). The same attachment site was also found in the nuclei of cultured chicken fibroblasts (Kalandadze et al., 1990). Hence at least within the domain of chicken o~-globin genes, the specific interaction of DNA with the nuclear matrix could be divided into permanent (preserved in non-active nuclei) and transient (or function-dependent) one. The major question was whether this is true for the whole genome. To answer this question we carried out renaturation experiments. In these experiments, trace amounts of either total DNA or runDNA from erythroblasts or erythrocytes were reannealed in the reactions driven by vast excess of either erythrocyte or erythroblast nmDNA (Fig. 2). The most important observation was that in both renaturation reactions at Cot values sufficient for complete renaturation of homologous nmDNA probes, total DNA probe reannealed only partially (25% unique sequences in reaction driven by erythroblast nmDNA and 10% of unique sequences in the reaction driven by erythrocyte nmDNA). This means that both nmDNA fractions are composed of specific subsets of unique sequences that are less complex than total DNA. The obvious conclusion that followed from the above observation is that nuclear matrix attachment sites have specific positions in the genome. Otherwise nmDNA preparations composed of DNA sequences surroun-
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ding the attachment sites would not differ from the total DNA. Similar considerations led to the conclusion that erythrocyte nmDNA is a subfraction of erythroblast nmDNA. Indeed, the latter did not reanneal completely in a reaction driven by erythrocyte nmDNA while both nmDNA probes reannealed completely in a reaction driven by erythroblast nmDNA. Hence, erythroblast nmDNA is composed of two subfractions: permanently attached to the nuclear matrix DNA (erythrocyte nmDNA) and DNA transiently associated with the nuclear matrix only in active nuclei. Hence, renaturation experiments demonstrated the significance of observations initially made in the experhnents with the domain of chicken o~-globin genes. Summarizing these data we can answer the first of the two questions mentioned at the beginning of this section and conclude that loop ends certainly have specific positions in the genome.
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Numerous attempts to answer the second of our questions, i.e. to fred out whether specific sequences are involved into organization of high-salt resistant attachment sites were not successful (see Georgiev et al.~ 1991) The most obvious approaches cloning and sequencing of short nuclear matrix DNA fragments. Unfortunately, the
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experiments of this type carried out in our and other laboratories (Jackson et al., 1984; Kuo, 1982; Georgiev et al., 1991) did not reveal specific sequence elements common for all or at least for a significant part of DNA fragments permanently attached to the nuclear matrix. However, most of these fragments contained some imperfect intemal repeats and recognition sites for different sequence-specific DNA-binding proteins. Current hypothesis suggested by Cook considers the high-salt resistant association of DNA with the nuclear matrix as a combination of a number of relatively weak individual interactions. Indeed, the attempts to pinpoint exact position of high-sah resistant attachment site by successive nuclease treatment never produced clear results. Usually rather large (several Kbp long) DNA fragments were clearly bound to the nuclear matrix, while none of their smaller parts could be identified with the attachment site. For example in our experiments the 1.7 kb long DNA fragment located ca. 3 Kbp upstream to the chicken pi-globin gene was found to be permanently attached to the nuclear matrix. When it was divided into four parts (0.5 kb long or less), all of them gave approximately the same signal when hybridized to the nuclear matrix DNA. It is not clear presently what weak interactions participate in organization of permanent attachment sites. At least some of them might be the interactions of DNA with sequence-specific DNA-binding regulatory protein factors. Indeed, a number of recognition sequences for different protein factors was found within all pans of the above mentioned 1.7 kb fragment of chicken DNA (De Moura-Gallo et al., 1991). Among other possibilities one can mention interactions with DNA-topoisomerase II that seem to be a major component of chromosomal scaffold (see below). SEQUENCE SPECIFICITY OF D N A INTERACTION WITH THE NUCLEAR (CHROMOSOMAL) SKELETON REVEALED IN THE EXPER/MENTS UTILIZING THE US-EXTRACTION METHOD OF NUCLEAR MATRIX ISOLATION AND IN VITRO D N A BINDING TO ISOLATED NUCLEAR
MATRIX (MARASSAY) Extraction of nuclei with concentrated salt solution used in the traditional method of nuclear matrix isolation was criticized as a source of different artifacts (rearrangement of nuclear material, precipitation of transcription complexes etc.). Thus several other procedures for isolation of nuclear matrix-bound DNA were developed. The best known is probably a US-extraction protocol designed by Laemmli and collaborators (Mircovich et al., 1984). According to this protocol, isolated nuclei were preextracted with mild ionic detergent (lithium diiodosalicilate) to remove the histones and then DNA was cleaved with restriction enzymes. Subsequent separation of scaffold-bound DNA from cleaved-off DNA was carried out by several washes in restriction buffer. One should mention that DNA interactions with the nuclear matrix revealed by this procedure are not necessarily high-salt resistant. Nevertheless, the scaffold-bound DNA fragments isolated by LIS-extraction procedure were found to constitute a specific subset of genomic DNA (Mircovich et al., 1984; Gasser and Laemmli, 1986). The same subset of restriction fragments of cloned genomic DNA was found to interact in a specific fashion with isolated nuclear matrices (even if they were isolated by high salt extraction procedure) (Cockerill and Garrard, 1986). Hence it is not clear whether the interactions of DNA with the nuclear matrix revealed by
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LIS-extraction method represent the pre-existing attachment sites or originate de novo during treatment of LIS-extracted nuclei with restriction enzymes. The important fact is that DNA-sequence elements responsible for these interactions, known as SARs (Mirkovich et al., 1984) or MARs (Cockerill and Garrard, 1986), were shown to have clear functional significance. The most important observation was that these elements were necessary for the function of yeast autonomously replicating sequences (ARS) (Amati and Gasser, 1988). Becides, it was reported (Phi-Van et al., 1990) that MARs ensure position-independent expression of reporter genes integrated into genome of eukaryotic cells. This may be particularly hnportant for understanding the domain organization of the genome as the reporter gene flanked by two MARs can be considered as an artificial mini-domain. The important question is whether there is any correlation between MARs and matrix-attactunent sites mapped by conventional methods. Initial observations stressed the difference between the interactions revealed by LIS-extraction and high-salt extraction (Mircovich et al., 1984). Indeed in active genome areas, the high-salt extraction procedure revealed interactions of transcribed sequences with the nuclear matrix while MARs (SARs) are specific DNA sequence elements and their distribution along the DNA chain does not depend on transcription or, more generally, on functional activity of the genome. However, in repressed avian erythrocyte nuclei (Razin et al., 1991) mad in non-transcribed regions of the genome in normal active nuclei (Mircovich et al., 1984, 1986) either one of the experimental protocols used (LIS-extraction, high salt-extraction and in vitro binding) indicated that the same regions were both attached to the nuclear matrix and possessed MARs. The distribution of MARs has recently been mapped within the large areas of the genome (in human and Drosophila ceils). The average distance in-between MARs in these areas turned out to be of the same order as the average size of above-discussed DNA loops (Mircovich, 1986). Hence it is likely that MARs participate in organization of permanent sites of DNA attachment to the nuclear matrix. DISTINCTION OF GENOMIC DOMAINS BY THE TOPO-II CLEAVAGE REACTION
In previous sections we have discussed the DNA sequences involved in interactions with the nuclear matrix. It is equally interesting to characterize the nuclear matrix proteins that interact with the borders of genomic domains. Little is known about these proteins. However, some data indicate that one of them is DNA-topoisomerase II. In particular it was shown that: (i) topoisomerase 1-I is one of the major components of the nuclear matrix and metaphase chromosomal scaffold (Berrios and Osheroff, 1985; Eamshaw and Heck, 1985); (ii) topoisomerase II is necessary for condensation of chromosomes at the onset of mitosis (Adachi et al., 1991); and (iii) MARs include putative recognition sites for topoisomerase II (Cockerill and Garrard, 1986). We have previously shown that MARs bound in vitro to isolated nuclear matrix could be cleaved by treatment of the complexes with VM26, a specific inhibitor of topoisomerase II known to fix intermediate products of cleavage/religation reaction catalyzed by this enzyme (Vassetzky et al., 1989). This result indicates that insoluble (matrix-bound) DNA topoisomerase II participates in interaction of MARs with the
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nuclear matrix. This does not exclude the possibility of the involvement of other proteins in the interaction (e.g. MAR-binding protein described by yon Kries et al. (1991)). It is important to stress that topoisomerase II is at least one of the proteins that interact with DNA at the bases of DNA loops. This £mding opens a possibility of excision and subsequent isolation of individual loops (see scheme in Fig. 3).
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Fig. 3. The scheme of experiments on excision of DNA loops by endogenous matrix-associated topoisomerase II. It is well-known that treatment of living cells with inhibitors of topoisomerase
II (VM26, VP16) fixes the intermediate complexes of topoisomerase 11 with DNA. Subsequent destruction of the enzyme in the course of cell lysis and DNA purification results in appearance of double-stranded DNA breaks at the sites of DNA interaction with topoisomerase II. We have analyzed the size distribution of DNA fragments excised from the genome by topoisomerase 11 cleavage and found it to be quite similar to the size distribution of the above discussed DNA loops (Razin et al. 1991b). Iaa full agreement with the model (Fig. 3), each excised DNA fragment was found to possess a protein molecule (presumably topoisomerase I / a t 5'-ends of both chains while 3'-termini were easily accessible to exonucleases (Razin et al., 1991a). Finally, we have shown that the ends of the excised DNA fragments have specific positions in the genome. In chicken cells we were able to identify unique excised DNA fragment containing the whole domain of o~-globin genes. Most hnportant, the upstream end of this fragment coincided with the previously mapped permanent site of DNA attachment to the nuclear matrix (Razin, 1986; Razin et al., 1991a). Subsequent analysis also revealed the presence of two MARs and numerous in vitro
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topoisomerase II cleavage sites within this area (Razin et al., 1991b). Thus, it seems that in vivo DNA cleavage with the matrix-bound DNA topoisomerase II can be used for excision of genomic domains. The latter can be then cloned in YAC vectors and organization of individual domains can be studied. The neighboring domains can be easily identified by cross-hybridization with the cloned nuclear matrix DNA fragments. In this way a long-distance physical map of the whole genome can be constructed. At the same time problems arise with the excision of DNA loops by topoisomerase II cleavage reaction. It is clear that significant (probably major) part of nuclear DNA topoisomerase II is located outside the chromosomal scaffold. This soluble enzyme will interact with any accessible (i.e. nucleosome-free) region in DNA. Indeed it was found by Reitman and Felsenfeld (1990) that under certain experimental conditions in erythroid ceils topoisomerase II introduce a number of breaks within the cluster of ~-globin genes at the positions coinciding with the positions of DNase I hypersensitive sites. Natttrally, the breaks of this type will decrease the specificity of excision of the domains. However, fortunately the soluble enzyme (even associated with DNA) could be easily extracted from the nuclei by medium or concentrated salt solution. At the same time the integrity of DNA loops (i.e. interaction of loop ends with the nuclear matrix) is not affected by such treatment. Hence, preextraction of nuclei with medium-concentrated salt solution before VM26 treatment or incubation of high-salt preextracted nuclei (nucleoids) in a media containing VM26 will probably solve the above problems and will significantly increase the specificity of cleavage in respect to the domain ends. The experhnents aimed to check this modification of domain excision protocol are presently in progress in our laboratory.
CONCLUSION We would like to return to the problem of correlations between functional and structural organization of the genome in the nuclei. The important question is: "What kind of functional genomic domains (if any) are defined by permanent attachment sites?" The presently available experimental data made it possible to say that first of all this are replication units. Indeed, we have found that replication origins are permanently attached to the nuclear matrix (Razin et al., 1986). This observation is in good agreement with the results of other authors (for review see Razin et al., 1990). This also explains the correlation between the average loop length and the average length of replicons in ceils of different species (Buongiorno et al., 1982). Average loop size determined by analysis of nucleoid sedimentation reflects predominantly the length of the loops defined by permanent attachment sites. Indeed, transient associations of transcriptionally active genes with the nuclear matrix usually ensure attachment of rather large genomic area (probably of the whole domain). This area will be simply excluded from the analysis as the sedimentation properties of nucleoids should clearly be affected mostly by the status (relaxed-supercoiled) of the long loops. Involvement of permanent sites of DNA attachment to the nuclear matrix in spatial organization of replicons does not exclude the possibility that they also define
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the transcriptional domains. The latter are considered here not as individual transcription units but rather as areas where the function of several transcription units (genes) can be controlled by the same regulatory mechanism. In this respect it is noteworthy that DNase I-sensitive domain composed of three functionally related chicken t~-globin genes was found to be located within an individual loop (Razin et al., 1991a). Analysis of spatial organization of other transcriptional domains will show whether this observation has general importance. REFERENCES Adachi Y., Luke M. and Laemmli U.K. (1991) Chromosome assembly in vitro: topoisomerase II required for condensation. Cell 64: 137-148. Amati B.B. and Gasser S.M. (1988) Chromosomal ARS and CEN elements bind specifically to the yeast nuclear scaffold. Cell 54: 967-978. Basler J., Hastie N.D., Pietras D., Matsui S.I., Sandberg A. and Berezney R. (1981) Hybridization of nuclear matrix attached DNA fragments. Biochemistry 20: 6921-6929. Berezney R. and Coffey D.S. (1976) The nuclear protein matrix: isolation, structure and functions. Adv. Enzyme Reg. 14:63-100 Berrios M., Osheroff N. and Fischer P.A. (1985) bl siru localization of DNA topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix fraction. Proc. Natl. Acad. Sci. USA 82:4142-4146 Buongiomo-Nardelli M., Gioacchino M., Carri M.T. and Marilley M. (1982) A relationship between replicon size and supercoiled loop domains in the eukaryotic genome. Nature 298: 100-102. Cockerill P.N. and Garrard W.T. (1986) Chromosomal loop anchorage of the kappa irmnunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell 44:273-282 Cook P.R. and Brazell I.A. (1980) Mapping sequences in loops of nuclear DNA by their progressive detachment from the nuclear cage. Nucl. Acids Res. 8: 2895-2906. Cook P.R., Lang J., Hayday A., Lania L., Fried M., Chiswell D.J. and Wyke A. (1982) Active viral genes in transformed cells lie close to the nuclear cage. EMBO J. i: 447-452. Cook P.R. (1989) The nucleoskeleton and the topology of transcription. Eur. J. Biochem. 185: 487-501. Eamshaw W.C. and Heck M.M.S. (1985) Localization of topoisomerase II in mitotic chromosomes. J. Cell Biol. 100: 1716-1725. De Moura Gallo C.V., Vassetzky Y.S., Recillas Targa F., Georgiev G.P., Scherrer K. and Razin S.V. (1991) The presence of sequence-specific protein binding sites correlate with replication activity and matrix binding in a 1.7 Kb-long DNA fragment of the chicken a-globin gene domain. Biochem. Biophys. Res. Commmun. 179: 512-519. Gasser S.M. and Laemmli U.K. (1986) The organization of chromatin loops: characterization of a scaffol attachment site. EMBO J. 5: 511-518. Georgiev G.P., Vassetzlcy Y.S., Luchnik A.N., Chemokhvostov V.V. and Razin S.V.
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(1991) Nuclear skeleton, DNA domains and control of replication and transcription. Eur. J. Biochem. 200: 613-624. Gross D.S. and Garrard W.T. (1988) Nuclease hypersensitive sites in chromatin. Ann. Rev. Biochem. 57: 159-197. Grosveld F., Van Assendelft B.G., Greaves D.R. and Kollias G. (1987) Position independent, high-level expression of the human ~-globin gene in transgenic mice. Cell 51: 975-985. Hancock R. and Huges M.E. (1982) Organization of DNA in the eukaryotic nucleus. Biol. Cell. 44: 201-212. Jackson D.A., Cook P.R. and Patel S.B. (1984) Attachment of repeated sequences to the nuclear cage. Nucl. Acids Res. 12: 6709-6726. Kalandadze A.G., Bushara S.A., Vassetzky Y.S. and Razin S.V. (1990) Characterization of DNA pattern in the site of permanent attachment to the nuclear matrix located in the vicinity of replication origin. Biochem. Biophys. Res. Commun. 188: 9-15. Kuo M.T. (1982) Analysis of DNA attached to the chromosome scaffold. J. Cell Biol. 93: 278-284. Matsumoto L.H. (1981) Enrichment of satellite DNA on the nuclear matrix of bovine cells. Nature 294: 481-482. Mirkovich J., Mirault M.E. and Laemmli U.K. (1984) Organization of the higher order chromatin loops: specific DNA attachment sites on nuclear scaffold. Cell 39: 223-232. Mirkovich J., Spierer P. and Laemmli U.K. (1986) Genes and loops in 320,000 base pairs of the Drosophila melanogaster chromosome. J. Mol. Biol. 190: 255-258. Pardoll D.M. and Vogelstein B. (1980) Sequence analysis of nuclear matrix-associated DNA from rat liver. Exp. Cell. Res. 28: 466-470. Phi-Van L., Von Kries J.P., Ostertag W. and Stratling W.H. (1990) The chicken lyzozyme 5' matrix attaclunent region increases transcription from a heterologous promoter in heterologous ceils and dampens position effects on the expression of transfected genes. Mol. Ceil. Biol. 10: 2302-2307. Razin S.V., Rzheshovska-Volni I., Moreau J. and Scherrer K. (1985) Localization of DNA attacment site to the nuclear matrix within the domain of chicken (x-globn genes in functionally actve and inactive nuclei. Moleklamaya Biologia USSR. 19:456-466 (in Russian). Razin S.V. (1987) DNA interaction with the nuclear matrix and spatial organisation of replication and transcription. BioEssays 6: 19-23. Razin S.V., Kekelidze M.G., Lukanidin E.M., Scherrer K. and Georgiev G.P. (1986) Replication origins are attached to the nuclear skeleton. Nucl. Acids Res. 14: 8189-8207. Razin S.V., Mantieva V.L. and Georgiev G.P. (1978) DNA adjacent to the attachment points of DNP fibril to chromosomal axial structure is enriched in reiterated base sequences. Nucl. Acids Res. 5: 4737-4751. Razin S.V., Mantieva V.L. and Georgiev G.P. (1979) The similarity of DNA sequences remaining bound to scaffold upon nuclease treatment of interphase nuclei and metaphase chromosomes. Nucl. Acids Res. 7" 1713-1735.
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Razin S.V., Petrov P. and Hancock R. (1991) Unique localisation of the ct-globin gene cluster within one of the 20-300 kb DNA fragments released by cleavage of chicken chromosomal DNA at topoisomerase II sites in vivo: evidence that the fragments are DNA loops or domains. Proc. Natl.Acad. Sci. USA, in press. Razin S.V., Vassetzky Y.S. and Hancock R. (1991) Nuclear matrix attachment region and topoisomerase II binding and reaction sites in the vicinity of a chicken DNA replication origin. Biochem. Biophys. Res. Commun. 177: 265-270. Razin S.V., Vassetskii Y.S. and Georgiev G.P. (1990) Replication origins of higher eukaryotes - a minireview. Biomed. Sci. 1: 18-22. Reeves R. (1984) Transcriptionally active chromatin. Biochhn. Biophys. Acta. 782: 343 -393. Reitman M. and Felsenfeld G. (1990) Developmental regulation of topoisomerase II sites and DNase 1-hypersensitive sites in the chicken ~-globin locus. Mol. Cell. Biol. 10: 2774-2786. Von Kries J.P., Buhrmeister H. and Stratling W.H. (1991) A matrix/scaffold attachment region binding protein: identification, purification and mode of binding. Cell 64: 123-135.
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DOMAIN ORGANIZATION OF EUKARYOTIC GENOME
S.V. Razin and Y.S. Vassetzky Institute of Biology of the Gene, USSR Academy of Sciences, 34/5 Vavilov st., Moscow 117334, USSR
INTRODUCTION It is a well-established fact that transcription of eukaryotic genes is regulated at several levels. One of these levels is represented by mechanisms that operate within relatively large genomic domains. Active domains are preferentially sensitive to DNase I (for review see Gross and Garrard, 1988). They could be several dozens kb long and usually include a group of functionally related genes (e.g. globin genes). The borders of the domains are quite sharp. Recent fmdings indicate that transcriptional status of a whole domain can be determined by a so-called dominant control region (DCR). The best known example is the dominant control region of the domain of human ~-globin genes (Grosveld et al., 1987; Groodin et al., 1989). A relatively short region located in the upstream area of this domain drastically influences the functional status of a genomic region that is more than 100 Kbp long (in particular: transcriptional status of this region, sensitivity to DNase I, methylation pattern and replication thning). Existence of functional domains in eukaryotic genome raises a question whether these domains correspond to some stmctural units in chromatin. There are at least three levels of DNA packaging in chromatin, namely nucleosomal level, solenoid level and DNA loops level (for review see Reeves, 1984). Only the structural units of the third level of DNA compactization (i.e. DNA loops anchored at the chromosomal skeleton) have the size comparable with the size of the above discussed functional genomic domains. The DNA loops were first recognized when sedimentation properties of high-salt extracted nuclei were studied (Cook and BrazeU, 1980). It turned out that after extraction of histones the DNA was topologically constrained and could be either supercofled or relaxed depending on some external parameters. The average size of constrained domains determined by several independent methods was found to be of the order of 100 kb (for review see Hancock and Huges, 1982). An attractive hypothesis has been suggested that individual loops correspond to the functional genomic domains (Cook, 1989) In the frame of this hypothesis it was easy to explain how the preferential sensitivity to DNase I could be maintained within the limited though large areas of the genome and why the borders of the sensitive regions are rather sharp. The most simple suggestion appeals to the possibility of reversible distraction of solenoid level of DNP packaging within the individual loops. All the above mentioned considerations draw particular attention to the problem of sequence specificity of DNA interaction with the chromosomal skeletal elements. Indeed, if loops correspond to functional genomic domains, one could expect that borders of the loops have specific positions on the DNA chain and and are located 0309-1651/92/080697-12/$03.00/0
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between the functional units of the genome. We shall discuss the results of experiments aimed to check this predictions. SEQUENCE SPECIFICITY OF DNA INTERACTIONWITH THE NUCLEAR(CHROMOSOMAL) SKELETON REVEALED IN THE EXPERIMENTS UTIIJZING HIGH SALT EXTRACTION PROTOCOL OF NUCLEAR MATRIX ISOLATION
The important feature of large DNA loops attached to the chromosomal scaffold that should be outlined here is the resistance of the whole structure to the extraction with concentrated salt solution. The remarkable stability of DNA interaction with chromosomal scaffold (that in interphase nuclei constitutes part of more complex structure known as nuclear matrix; Berezney and Coffey, 1976) opened a simple possibility of isolation of DNA fragments located at basements of chromatin loops. For isolation of loop ends one simply has to preextract nuclei with concentrated or medium salt solution, than to introduce double-stranded breaks into DNA chain (limited digestion with restriction enzymes or limited digestion with sequence-nonspecific nucleases) and finally to separate scaffold-bound and cleaved-off DNA by several washes with concentrated salt solution (Cook and Brazell, 1980; Pardoll and Vogelstein, 1980; Razin et al., 1979). Different modifications of this approach were widely used for isolation of the so-called nuclear matrix DNA (nmDNA). The latter can be defined as a preparation of DNA fragments that remains bound to the residual nuclear structures after high-salt extraction of nuclease-treated nuclei. The variations in experimental procedures used by different authors usually involved utilization of different salts (NaCI, KCI, (NH4)2SO4, etc.) and different concentrations of salt for preextraction and final extraction as well as utilization of different enzymes for DNA cleavage (for review see Razin, 1987). After isolation of nmDNA, one obviously tried to answer two distinct, though related questions: I. Do the loop ends have specific positions in the genome? and II. Are there specific DNA sequences located at the basements of DNA loops? In our initial experiments (Razin et al., 1978, 1979) the renaturation properties of nmDNA were studied. This DNA was found to be moderately enriched in repetitive sequences. This observation was later confumed by some authors (Hancock and Huges, 1982; Jackson et al., 1984; Matsumoto, 1981) while the others did not observe any specificity of runDNA (Basler et al., 1981). Possible reasons of these contradictions have been discussed previously (Razin et al., 1985; Razin, 1987). More disappointing, no particular DNA anchoring sequence was found either in our experiments or in experiments of other authors. Even though enriched in repetitive sequences, the nmDNA still contained a significant portion of unique sequences. The spectrum of repeats represented in nmDNA was also rather broad. More meaningful results were obtained in the experiments ahned to find the relative positions of different genes within DN.A loops. These experiments initially designed by Cook and Brazell (1980) were later carded out in a number of laboratories. In most of the papers published so far, a correlation between the transcriptional status of a gene and its attaclmaent to the nuclear matrix was observed. The active genes were generally found in the nmDNA while the non-active ones in the cleaved off fraction (for review see Razin, 1987). The most convincing results
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were probably obtained in the experiments with hormone-induced premature cell differentiation. The cell type specific genes that started expression in differentiated cells were associated with the nuclear matrix in these cells and were not associated with the nuclear matrix in maternal (non-differentiated) cells.
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Fig. 1. Detection of specific regions in the chicken tx-globin domain that interact with erythrocyte and erythroblast nmDNA (Razin et al., 1985). Left panel: identification of attachment sites in chicken erythroblast nuclei. Restriction fragments of recombinant clones were transferred onto nylon filters and hybridized with either nick-translated total DNA (a-f) or erythroblast nmDNA probe (a'-f'). The scheme below indicates the regions of the domain of chicken 0t-globin genes that are highly (black) or moderately (shaded) enriched in nmDNA as compared to total DNA. Circles-filled areas correspond to repetitive sequences. Arrows indicate recognition sites for restriction endonucleases used. Right panel: identification of attachment sites in chicken erythrocyte nuclei. Restriction fragments of recombinant clones were transferred onto nylon filters and hybridized with either nick-translated total DNA (a-e) or erythrocyte nmDNA probe (a'-e'). The scheme is similar to that in the left panel. The scheme below the panels summarizes the data on distribution of attachment sites in the both ceil types.
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The positive correlation between expression of viral genomes integrated into the genome of host cells and their attachment to the nuclear matrix of these cells was also reported (Cook et al., 1982). When the large cloned areas of the eukaryotic genome became available, the positions of preferential sites of DNA interaction with the nuclear matrix have been mapped within several such areas (Razin et al., 1986, 1990) and the positive correlation between DNA attachment to the nuclear matrix mad its transcription has also been observed. All the above-mentioned results could h a r d y be assimilated by the initial model based on the assumption that there was a correlation between DNA loop organization and domain organization of the genome. Indeed, all the specific interactions of DNA with nuclear matrix seemed to be related to some functional processes and patterns of these interactions were different in the cells of different lineages. At the same time a new model of DNA loop organization based on function-dependent association of DNA with the nuclear matrix also could not explain all the experimental data, for example a correlation of DNA loops size with the average sizes of replicons in the cells of different species (Buongiomo et al., 1982). Several years ago we suggested that there is a special group of "structural" attachment sites that are different from transcription-related attachment sites and that the former defined the borders of genomic domains. To check the idea we carried out experiments with inactive nuclei, e.g. mature sperm nuclei or repressed nuclei of avian erythrocytes. In these nuclei, all the function-dependent interactions of DNA with the nuclear matrix should disappear and "structural" interactions, should be easily visible. We have mapped preferential positions of sites of DNA attachment to the nuclear matrix in the domain of chicken ct-globin genes in chicken erythroblasts, erythrocytes and mature sperm nuclei. The results of our experiments (Fig. 1) supported the initial suggestion. While in erythroblast nuclei the whole transcriptionally active area was attached to the nuclear matrix, in erythrocyte and sperm nuclei only one major attachment site was found in the upstream region of the domain (Razin et al., 1985; Razin, 1987; Kalandadze et al., 1990). The same attachment site was also found in the nuclei of cultured chicken fibroblasts (Kalandadze et al., 1990). Hence at least within the domain of chicken o~-globin genes, the specific interaction of DNA with the nuclear matrix could be divided into permanent (preserved in non-active nuclei) and transient (or function-dependent) one. The major question was whether this is true for the whole genome. To answer this question we carried out renaturation experiments. In these experiments, trace amounts of either total DNA or runDNA from erythroblasts or erythrocytes were reannealed in the reactions driven by vast excess of either erythrocyte or erythroblast nmDNA (Fig. 2). The most important observation was that in both renaturation reactions at Cot values sufficient for complete renaturation of homologous nmDNA probes, total DNA probe reannealed only partially (25% unique sequences in reaction driven by erythroblast nmDNA and 10% of unique sequences in the reaction driven by erythrocyte nmDNA). This means that both nmDNA fractions are composed of specific subsets of unique sequences that are less complex than total DNA. The obvious conclusion that followed from the above observation is that nuclear matrix attachment sites have specific positions in the genome. Otherwise nmDNA preparations composed of DNA sequences surroun-
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ding the attachment sites would not differ from the total DNA. Similar considerations led to the conclusion that erythrocyte nmDNA is a subfraction of erythroblast nmDNA. Indeed, the latter did not reanneal completely in a reaction driven by erythrocyte nmDNA while both nmDNA probes reannealed completely in a reaction driven by erythroblast nmDNA. Hence, erythroblast nmDNA is composed of two subfractions: permanently attached to the nuclear matrix DNA (erythrocyte nmDNA) and DNA transiently associated with the nuclear matrix only in active nuclei. Hence, renaturation experiments demonstrated the significance of observations initially made in the experhnents with the domain of chicken o~-globin genes. Summarizing these data we can answer the first of the two questions mentioned at the beginning of this section and conclude that loop ends certainly have specific positions in the genome.
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Lg Cot Fig. 2. Corenaturation of tracer amounts of highly labeled total DNA and nmDNA probes from chicken erythrocytes and erythroblasts with the excess of unlabeled erythroblast (A) and erythrocyte (B) nmDNA (Razin et al., 1986). The origin of labeled probe is indicated near each curve. Broken line shows renaturation of total DNA in a reaction driven by total DNA. O R G A N I Z A T I O N O F T H E H I G H S A L T R E S I S T A N T Aq'TACI-IEMENT SITES
Numerous attempts to answer the second of our questions, i.e. to fred out whether specific sequences are involved into organization of high-salt resistant attachment sites were not successful (see Georgiev et al.~ 1991) The most obvious approaches cloning and sequencing of short nuclear matrix DNA fragments. Unfortunately, the
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experiments of this type carried out in our and other laboratories (Jackson et al., 1984; Kuo, 1982; Georgiev et al., 1991) did not reveal specific sequence elements common for all or at least for a significant part of DNA fragments permanently attached to the nuclear matrix. However, most of these fragments contained some imperfect intemal repeats and recognition sites for different sequence-specific DNA-binding proteins. Current hypothesis suggested by Cook considers the high-salt resistant association of DNA with the nuclear matrix as a combination of a number of relatively weak individual interactions. Indeed, the attempts to pinpoint exact position of high-sah resistant attachment site by successive nuclease treatment never produced clear results. Usually rather large (several Kbp long) DNA fragments were clearly bound to the nuclear matrix, while none of their smaller parts could be identified with the attachment site. For example in our experiments the 1.7 kb long DNA fragment located ca. 3 Kbp upstream to the chicken pi-globin gene was found to be permanently attached to the nuclear matrix. When it was divided into four parts (0.5 kb long or less), all of them gave approximately the same signal when hybridized to the nuclear matrix DNA. It is not clear presently what weak interactions participate in organization of permanent attachment sites. At least some of them might be the interactions of DNA with sequence-specific DNA-binding regulatory protein factors. Indeed, a number of recognition sequences for different protein factors was found within all pans of the above mentioned 1.7 kb fragment of chicken DNA (De Moura-Gallo et al., 1991). Among other possibilities one can mention interactions with DNA-topoisomerase II that seem to be a major component of chromosomal scaffold (see below). SEQUENCE SPECIFICITY OF D N A INTERACTION WITH THE NUCLEAR (CHROMOSOMAL) SKELETON REVEALED IN THE EXPER/MENTS UTILIZING THE US-EXTRACTION METHOD OF NUCLEAR MATRIX ISOLATION AND IN VITRO D N A BINDING TO ISOLATED NUCLEAR
MATRIX (MARASSAY) Extraction of nuclei with concentrated salt solution used in the traditional method of nuclear matrix isolation was criticized as a source of different artifacts (rearrangement of nuclear material, precipitation of transcription complexes etc.). Thus several other procedures for isolation of nuclear matrix-bound DNA were developed. The best known is probably a US-extraction protocol designed by Laemmli and collaborators (Mircovich et al., 1984). According to this protocol, isolated nuclei were preextracted with mild ionic detergent (lithium diiodosalicilate) to remove the histones and then DNA was cleaved with restriction enzymes. Subsequent separation of scaffold-bound DNA from cleaved-off DNA was carried out by several washes in restriction buffer. One should mention that DNA interactions with the nuclear matrix revealed by this procedure are not necessarily high-salt resistant. Nevertheless, the scaffold-bound DNA fragments isolated by LIS-extraction procedure were found to constitute a specific subset of genomic DNA (Mircovich et al., 1984; Gasser and Laemmli, 1986). The same subset of restriction fragments of cloned genomic DNA was found to interact in a specific fashion with isolated nuclear matrices (even if they were isolated by high salt extraction procedure) (Cockerill and Garrard, 1986). Hence it is not clear whether the interactions of DNA with the nuclear matrix revealed by
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LIS-extraction method represent the pre-existing attachment sites or originate de novo during treatment of LIS-extracted nuclei with restriction enzymes. The important fact is that DNA-sequence elements responsible for these interactions, known as SARs (Mirkovich et al., 1984) or MARs (Cockerill and Garrard, 1986), were shown to have clear functional significance. The most important observation was that these elements were necessary for the function of yeast autonomously replicating sequences (ARS) (Amati and Gasser, 1988). Becides, it was reported (Phi-Van et al., 1990) that MARs ensure position-independent expression of reporter genes integrated into genome of eukaryotic cells. This may be particularly hnportant for understanding the domain organization of the genome as the reporter gene flanked by two MARs can be considered as an artificial mini-domain. The important question is whether there is any correlation between MARs and matrix-attactunent sites mapped by conventional methods. Initial observations stressed the difference between the interactions revealed by LIS-extraction and high-salt extraction (Mircovich et al., 1984). Indeed in active genome areas, the high-salt extraction procedure revealed interactions of transcribed sequences with the nuclear matrix while MARs (SARs) are specific DNA sequence elements and their distribution along the DNA chain does not depend on transcription or, more generally, on functional activity of the genome. However, in repressed avian erythrocyte nuclei (Razin et al., 1991) mad in non-transcribed regions of the genome in normal active nuclei (Mircovich et al., 1984, 1986) either one of the experimental protocols used (LIS-extraction, high salt-extraction and in vitro binding) indicated that the same regions were both attached to the nuclear matrix and possessed MARs. The distribution of MARs has recently been mapped within the large areas of the genome (in human and Drosophila ceils). The average distance in-between MARs in these areas turned out to be of the same order as the average size of above-discussed DNA loops (Mircovich, 1986). Hence it is likely that MARs participate in organization of permanent sites of DNA attachment to the nuclear matrix. DISTINCTION OF GENOMIC DOMAINS BY THE TOPO-II CLEAVAGE REACTION
In previous sections we have discussed the DNA sequences involved in interactions with the nuclear matrix. It is equally interesting to characterize the nuclear matrix proteins that interact with the borders of genomic domains. Little is known about these proteins. However, some data indicate that one of them is DNA-topoisomerase II. In particular it was shown that: (i) topoisomerase 1-I is one of the major components of the nuclear matrix and metaphase chromosomal scaffold (Berrios and Osheroff, 1985; Eamshaw and Heck, 1985); (ii) topoisomerase II is necessary for condensation of chromosomes at the onset of mitosis (Adachi et al., 1991); and (iii) MARs include putative recognition sites for topoisomerase II (Cockerill and Garrard, 1986). We have previously shown that MARs bound in vitro to isolated nuclear matrix could be cleaved by treatment of the complexes with VM26, a specific inhibitor of topoisomerase II known to fix intermediate products of cleavage/religation reaction catalyzed by this enzyme (Vassetzky et al., 1989). This result indicates that insoluble (matrix-bound) DNA topoisomerase II participates in interaction of MARs with the
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nuclear matrix. This does not exclude the possibility of the involvement of other proteins in the interaction (e.g. MAR-binding protein described by yon Kries et al. (1991)). It is important to stress that topoisomerase II is at least one of the proteins that interact with DNA at the bases of DNA loops. This £mding opens a possibility of excision and subsequent isolation of individual loops (see scheme in Fig. 3).
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Fig. 3. The scheme of experiments on excision of DNA loops by endogenous matrix-associated topoisomerase II. It is well-known that treatment of living cells with inhibitors of topoisomerase
II (VM26, VP16) fixes the intermediate complexes of topoisomerase 11 with DNA. Subsequent destruction of the enzyme in the course of cell lysis and DNA purification results in appearance of double-stranded DNA breaks at the sites of DNA interaction with topoisomerase II. We have analyzed the size distribution of DNA fragments excised from the genome by topoisomerase 11 cleavage and found it to be quite similar to the size distribution of the above discussed DNA loops (Razin et al. 1991b). Iaa full agreement with the model (Fig. 3), each excised DNA fragment was found to possess a protein molecule (presumably topoisomerase I / a t 5'-ends of both chains while 3'-termini were easily accessible to exonucleases (Razin et al., 1991a). Finally, we have shown that the ends of the excised DNA fragments have specific positions in the genome. In chicken cells we were able to identify unique excised DNA fragment containing the whole domain of o~-globin genes. Most hnportant, the upstream end of this fragment coincided with the previously mapped permanent site of DNA attachment to the nuclear matrix (Razin, 1986; Razin et al., 1991a). Subsequent analysis also revealed the presence of two MARs and numerous in vitro
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topoisomerase II cleavage sites within this area (Razin et al., 1991b). Thus, it seems that in vivo DNA cleavage with the matrix-bound DNA topoisomerase II can be used for excision of genomic domains. The latter can be then cloned in YAC vectors and organization of individual domains can be studied. The neighboring domains can be easily identified by cross-hybridization with the cloned nuclear matrix DNA fragments. In this way a long-distance physical map of the whole genome can be constructed. At the same time problems arise with the excision of DNA loops by topoisomerase II cleavage reaction. It is clear that significant (probably major) part of nuclear DNA topoisomerase II is located outside the chromosomal scaffold. This soluble enzyme will interact with any accessible (i.e. nucleosome-free) region in DNA. Indeed it was found by Reitman and Felsenfeld (1990) that under certain experimental conditions in erythroid ceils topoisomerase II introduce a number of breaks within the cluster of ~-globin genes at the positions coinciding with the positions of DNase I hypersensitive sites. Natttrally, the breaks of this type will decrease the specificity of excision of the domains. However, fortunately the soluble enzyme (even associated with DNA) could be easily extracted from the nuclei by medium or concentrated salt solution. At the same time the integrity of DNA loops (i.e. interaction of loop ends with the nuclear matrix) is not affected by such treatment. Hence, preextraction of nuclei with medium-concentrated salt solution before VM26 treatment or incubation of high-salt preextracted nuclei (nucleoids) in a media containing VM26 will probably solve the above problems and will significantly increase the specificity of cleavage in respect to the domain ends. The experhnents aimed to check this modification of domain excision protocol are presently in progress in our laboratory.
CONCLUSION We would like to return to the problem of correlations between functional and structural organization of the genome in the nuclei. The important question is: "What kind of functional genomic domains (if any) are defined by permanent attachment sites?" The presently available experimental data made it possible to say that first of all this are replication units. Indeed, we have found that replication origins are permanently attached to the nuclear matrix (Razin et al., 1986). This observation is in good agreement with the results of other authors (for review see Razin et al., 1990). This also explains the correlation between the average loop length and the average length of replicons in ceils of different species (Buongiorno et al., 1982). Average loop size determined by analysis of nucleoid sedimentation reflects predominantly the length of the loops defined by permanent attachment sites. Indeed, transient associations of transcriptionally active genes with the nuclear matrix usually ensure attachment of rather large genomic area (probably of the whole domain). This area will be simply excluded from the analysis as the sedimentation properties of nucleoids should clearly be affected mostly by the status (relaxed-supercoiled) of the long loops. Involvement of permanent sites of DNA attachment to the nuclear matrix in spatial organization of replicons does not exclude the possibility that they also define
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the transcriptional domains. The latter are considered here not as individual transcription units but rather as areas where the function of several transcription units (genes) can be controlled by the same regulatory mechanism. In this respect it is noteworthy that DNase I-sensitive domain composed of three functionally related chicken t~-globin genes was found to be located within an individual loop (Razin et al., 1991a). Analysis of spatial organization of other transcriptional domains will show whether this observation has general importance. REFERENCES Adachi Y., Luke M. and Laemmli U.K. (1991) Chromosome assembly in vitro: topoisomerase II required for condensation. Cell 64: 137-148. Amati B.B. and Gasser S.M. (1988) Chromosomal ARS and CEN elements bind specifically to the yeast nuclear scaffold. Cell 54: 967-978. Basler J., Hastie N.D., Pietras D., Matsui S.I., Sandberg A. and Berezney R. (1981) Hybridization of nuclear matrix attached DNA fragments. Biochemistry 20: 6921-6929. Berezney R. and Coffey D.S. (1976) The nuclear protein matrix: isolation, structure and functions. Adv. Enzyme Reg. 14:63-100 Berrios M., Osheroff N. and Fischer P.A. (1985) bl siru localization of DNA topoisomerase II, a major polypeptide component of the Drosophila nuclear matrix fraction. Proc. Natl. Acad. Sci. USA 82:4142-4146 Buongiomo-Nardelli M., Gioacchino M., Carri M.T. and Marilley M. (1982) A relationship between replicon size and supercoiled loop domains in the eukaryotic genome. Nature 298: 100-102. Cockerill P.N. and Garrard W.T. (1986) Chromosomal loop anchorage of the kappa irmnunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell 44:273-282 Cook P.R. and Brazell I.A. (1980) Mapping sequences in loops of nuclear DNA by their progressive detachment from the nuclear cage. Nucl. Acids Res. 8: 2895-2906. Cook P.R., Lang J., Hayday A., Lania L., Fried M., Chiswell D.J. and Wyke A. (1982) Active viral genes in transformed cells lie close to the nuclear cage. EMBO J. i: 447-452. Cook P.R. (1989) The nucleoskeleton and the topology of transcription. Eur. J. Biochem. 185: 487-501. Eamshaw W.C. and Heck M.M.S. (1985) Localization of topoisomerase II in mitotic chromosomes. J. Cell Biol. 100: 1716-1725. De Moura Gallo C.V., Vassetzky Y.S., Recillas Targa F., Georgiev G.P., Scherrer K. and Razin S.V. (1991) The presence of sequence-specific protein binding sites correlate with replication activity and matrix binding in a 1.7 Kb-long DNA fragment of the chicken a-globin gene domain. Biochem. Biophys. Res. Commmun. 179: 512-519. Gasser S.M. and Laemmli U.K. (1986) The organization of chromatin loops: characterization of a scaffol attachment site. EMBO J. 5: 511-518. Georgiev G.P., Vassetzlcy Y.S., Luchnik A.N., Chemokhvostov V.V. and Razin S.V.
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(1991) Nuclear skeleton, DNA domains and control of replication and transcription. Eur. J. Biochem. 200: 613-624. Gross D.S. and Garrard W.T. (1988) Nuclease hypersensitive sites in chromatin. Ann. Rev. Biochem. 57: 159-197. Grosveld F., Van Assendelft B.G., Greaves D.R. and Kollias G. (1987) Position independent, high-level expression of the human ~-globin gene in transgenic mice. Cell 51: 975-985. Hancock R. and Huges M.E. (1982) Organization of DNA in the eukaryotic nucleus. Biol. Cell. 44: 201-212. Jackson D.A., Cook P.R. and Patel S.B. (1984) Attachment of repeated sequences to the nuclear cage. Nucl. Acids Res. 12: 6709-6726. Kalandadze A.G., Bushara S.A., Vassetzky Y.S. and Razin S.V. (1990) Characterization of DNA pattern in the site of permanent attachment to the nuclear matrix located in the vicinity of replication origin. Biochem. Biophys. Res. Commun. 188: 9-15. Kuo M.T. (1982) Analysis of DNA attached to the chromosome scaffold. J. Cell Biol. 93: 278-284. Matsumoto L.H. (1981) Enrichment of satellite DNA on the nuclear matrix of bovine cells. Nature 294: 481-482. Mirkovich J., Mirault M.E. and Laemmli U.K. (1984) Organization of the higher order chromatin loops: specific DNA attachment sites on nuclear scaffold. Cell 39: 223-232. Mirkovich J., Spierer P. and Laemmli U.K. (1986) Genes and loops in 320,000 base pairs of the Drosophila melanogaster chromosome. J. Mol. Biol. 190: 255-258. Pardoll D.M. and Vogelstein B. (1980) Sequence analysis of nuclear matrix-associated DNA from rat liver. Exp. Cell. Res. 28: 466-470. Phi-Van L., Von Kries J.P., Ostertag W. and Stratling W.H. (1990) The chicken lyzozyme 5' matrix attaclunent region increases transcription from a heterologous promoter in heterologous ceils and dampens position effects on the expression of transfected genes. Mol. Ceil. Biol. 10: 2302-2307. Razin S.V., Rzheshovska-Volni I., Moreau J. and Scherrer K. (1985) Localization of DNA attacment site to the nuclear matrix within the domain of chicken (x-globn genes in functionally actve and inactive nuclei. Moleklamaya Biologia USSR. 19:456-466 (in Russian). Razin S.V. (1987) DNA interaction with the nuclear matrix and spatial organisation of replication and transcription. BioEssays 6: 19-23. Razin S.V., Kekelidze M.G., Lukanidin E.M., Scherrer K. and Georgiev G.P. (1986) Replication origins are attached to the nuclear skeleton. Nucl. Acids Res. 14: 8189-8207. Razin S.V., Mantieva V.L. and Georgiev G.P. (1978) DNA adjacent to the attachment points of DNP fibril to chromosomal axial structure is enriched in reiterated base sequences. Nucl. Acids Res. 5: 4737-4751. Razin S.V., Mantieva V.L. and Georgiev G.P. (1979) The similarity of DNA sequences remaining bound to scaffold upon nuclease treatment of interphase nuclei and metaphase chromosomes. Nucl. Acids Res. 7" 1713-1735.
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Razin S.V., Petrov P. and Hancock R. (1991) Unique localisation of the ct-globin gene cluster within one of the 20-300 kb DNA fragments released by cleavage of chicken chromosomal DNA at topoisomerase II sites in vivo: evidence that the fragments are DNA loops or domains. Proc. Natl.Acad. Sci. USA, in press. Razin S.V., Vassetzky Y.S. and Hancock R. (1991) Nuclear matrix attachment region and topoisomerase II binding and reaction sites in the vicinity of a chicken DNA replication origin. Biochem. Biophys. Res. Commun. 177: 265-270. Razin S.V., Vassetskii Y.S. and Georgiev G.P. (1990) Replication origins of higher eukaryotes - a minireview. Biomed. Sci. 1: 18-22. Reeves R. (1984) Transcriptionally active chromatin. Biochhn. Biophys. Acta. 782: 343 -393. Reitman M. and Felsenfeld G. (1990) Developmental regulation of topoisomerase II sites and DNase 1-hypersensitive sites in the chicken ~-globin locus. Mol. Cell. Biol. 10: 2774-2786. Von Kries J.P., Buhrmeister H. and Stratling W.H. (1991) A matrix/scaffold attachment region binding protein: identification, purification and mode of binding. Cell 64: 123-135.